Env Immunogens Research Articles

Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. Here, to precisely engineer bnAb boosting immunogens, we use molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identify Env mutations predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encode antibody affinity gains and select for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

HIV-1 envelope (Env) is the key target for antibodies (Abs) against the virus and thus an important HIV-1 vaccine component. Env is synthesized from a gp160 precursor with a signal peptide (SP) at its N-terminus. This study investigated the influence of the SP on Env antigenicity and immunogenicity. Env proteins from two HIV-1 isolates, AA05 and AC02, were analyzed as gp120 and gp160 in their native wild-type (WT) forms and as chimeras with swapped SPs (AA05-02 and AC02-05). The WT and chimeric Env were assessed for antigenicity and glycosylation using monoclonal antibodies (mAbs) and glycan probes. Immunogenicity was tested in mice using three vaccine types: gp120 protein, gp120 DNA+gp120 protein, and gp120 DNA+gp160 DNA. The recombinant AC02 gp120 protein was antigenically superior to AA05 as indicated by higher reactivity with most mAbs tested. When SPs were swapped, the antigenicity of the chimeric gp120s (AA05-02 and AC02-05) resembled that of the gp120s from which the SPs were derived; AA05-02 was similar to AC02 and vice versa. Glycan probe reactivity followed a similar pattern: AA05-02 and AC02 showed similar affinity to high-mannose specific mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02. These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to conserved epitopes on HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The animal models commonly used for HIV do not reliably produce a potent broadly neutralizing serum antibody response, with the exception of cows. Cows have previously produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models, other engineered immunogens were shown to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n = 4) with two regimens of V2-apex focusing Env immunogens to investigate whether antibody responses could be generated to the V2-apex on Env. Group 1 was immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 was immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows, respectively, and showed moderate breadth and potency. Potent and broad responses in this study developed much later than previous cow immunizations that elicited CD4bs bnAbs responses and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target more than one broadly neutralizing epitope on the HIV surface reveals the generality of elongated structures for the recognition of highly glycosylated proteins. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Arenavirus-Based Vectors Generate Robust SIV Immunity in Non-Human Primates.

Arenavirus-based vectors are being investigated as therapeutic vaccine candidates with the potential to elicit robust CD8 T-cell responses. We compared the immunogenicity of replicating (artPICV and artLCMV) and non-replicating (rPICV and rLCMV) arenavirus-based vectors expressing simian immunodeficiency virus (SIV) Gag and Envelope (Env) immunogens in treatment-naïve non-human primates. Heterologous regimens with non-replicating and replicating vectors elicited more robust SIV IFN-γ responses than a homologous regimen, and replicating vectors elicited significantly higher cellular immunogenicity than non-replicating vectors. The heterologous regimen elicited high anti-Env antibody titers when administered intravenously, with replicating vectors inducing significantly higher titers than non-replicating vectors. Intramuscular immunization resulted in more durable antibody responses than intravenous immunization for both vector platforms, with no difference between the replicating and non-replicating vectors. Overall, both replicating and non-replicating arenavirus vectors generated robust T- and B-cell-mediated immunity to SIV antigens in treatment-naïve non-human primates, supporting further evaluation of these vectors in a clinical setting for HIV therapy.

Viral Envelope Evolution in Simian-HIV-Infected Neonate and Adult-Dam Pairs of Rhesus Macaques.

We recently demonstrated that Simian-HIV (SHIV)-infected neonate rhesus macaques (RMs) generated heterologous HIV-1 neutralizing antibodies (NAbs) with broadly-NAb (bNAb) characteristics at a higher frequency compared with their corresponding dam. Here, we characterized genetic diversity in Env sequences from four neonate or adult/dam RM pairs: in two pairs, neonate and dam RMs made heterologous HIV-1 NAbs; in one pair, neither the neonate nor the dam made heterologous HIV-1 NAbs; and in another pair, only the neonate made heterologous HIV-1 NAbs. Phylogenetic and sequence diversity analyses of longitudinal Envs revealed that a higher genetic diversity, within the host and away from the infecting SHIV strain, was correlated with heterologous HIV-1 NAb development. We identified 22 Env variable sites, of which 9 were associated with heterologous HIV-1 NAb development; 3/9 sites had mutations previously linked to HIV-1 Env bNAb development. These data suggested that viral diversity drives heterologous HIV-1 NAb development, and the faster accumulation of viral diversity in neonate RMs may be a potential mechanism underlying bNAb induction in pediatric populations. Moreover, these data may inform candidate Env immunogens to guide precursor B cells to bNAb status via vaccination by the Env-based selection of bNAb lineage members with the appropriate mutations associated with neutralization breadth.

Safety and immunogenicity of a polyvalent DNA–protein HIV vaccine with matched Env immunogens delivered as a prime–boost regimen or coadministered in HIV-uninfected adults in the USA (HVTN 124): a phase 1, placebo-controlled, double-blind randomised controlled trial

An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. Both vaccine regimens were safe, warranting evaluation in larger trials. US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines.

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

Enhancing anti-viral neutralization response to immunization with HIV-1 envelope glycoprotein immunogens

An effective human immunodeficiency virus type I (HIV-1) vaccine that robustly elicits broadly neutralizing antibodies (bnAbs) against HIV-1 envelope glycoproteins (Envs) to block viral entry is still not available. Thus, identifying triggers for elicitation of different types of anti-HIV-1 Env antibodies by vaccination could provide further guidance for immunogen design and vaccine development. Here, we studied the immune response to HIV-1 Env immunogens in rabbits. We show that sequential immunizations with conformation-specific Env immunogens can elicit low titer but broad neutralization responses against heterologous, neutralization-resistant (tier 2/3) transmitted/founder (T/F) HIV-1 strains. More importantly, an mRNA vaccine candidate that could mediate the presentation of a cytoplasmic tail-deleted (ΔCT) HIV-1AD8 Env immunogen on virus-like particles significantly increased the neutralization response. This strategy shifted the type of elicited antibodies, decreasing the level of binding to soluble Envs while significantly increasing their overall viral neutralization activity. The breadth and potency of neutralizing response against heterologous, T/F HIV-1 strains significantly increased in a subset of rabbits. Efficient neutralization activity was associated with high cellular immune responses specific to HIV-1 Envs. These results help to understand the immune response to different immunization schemes and will allow developing new approaches to selectively manipulate the type of humoral immune response by specific vaccination.

Assessing immunogenicity barriers of the HIV-1 envelope trimer

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.

'Immunization during ART and ATI for HIV-1 vaccine discovery/development'.

Explore whether immunization with germline-targeting Env immunogens during ART, followed by ATI, leads to the identification of viral envelope glycoproteins (Envs) that promote and guide the full maturation of broadly neutralizing antibody responses. The HIV-1 envelope glycoprotein (Env) does not efficiently engage the germline precursors of broadly neutralizing antibodies (bnAbs). However, Env-derived proteins specifically designed to precisely do that, have been recently developed. These 'germline-targeting' Env immunogens activate naïve B cells that express the germline precursors of bnAbs but by themselves cannot guide their maturation towards their broadly neutralizing forms. This requires sequential immunizations with heterologous sets of Envs. These 'booster' Envs are currently unknown. Combining germline-targeting Env immunization approaches during ART with ATI could lead to the identification of natural Envs that are responsible for the maturation of broadly neutralizing antibody responses during infection. Such Envs could then serve as booster immunogens to guide the maturation of glBCRs that have become activated by germline-targeting immunogens in uninfected subjects.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3.As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

Stabilized HIV-1 envelope immunization induces neutralizing antibodies to the CD4bs and protects macaques against mucosal infection.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4+ T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine-induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

Augmenting glycosylation‐directed folding pathways enhances the fidelity of HIV Env immunogen production in plants

Heterologous glycoprotein production relies on host glycosylation‐dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco‐engineering to improve the production of a model HIV‐1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3‐fucosyltransferase and β1,2‐xylosyltransferase was used as an expression host to prevent plant‐specific complex N‐glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco‐engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell‐produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant‐produced and mammalian cell‐derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus‐vectored vaccines. This study demonstrates that engineering glycosylation‐directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

Characterization of a vaccine-elicited human antibody with sequence homology to VRC01-class antibodies that binds the C1C2 gp120 domain.

Broadly HIV-1–neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02–derived heavy chains paired with light chains expressing five–amino acid–long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line–targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans.

Targeting tonsillar B cells with IL-9 and IL-33 enhances the rhesus macaque humoral response to DNA/protein-based HIV envelope vaccine

Abstract Development of an effective HIV vaccine is dependent on the quantity and quality of HIV Envelope specific antibody at the mucosal sites. Priming of the oral mucosa is a promising approach to generate mucosal antibody at HIV entry sites. Our recent work in mice has demonstrated the ability of IL-9 and IL-33, known regulators of mucosal immunity, to promote robust development of HIV Env-specific IgG; in addition they increase the breadth, magnitude, and durability of the Env-specific IgG response when combined with the DNA/protein HIV Env immunogen platform VC10014, which elicits Tier 2 neutralizing antibody in rhesus macaques. We hypothesized that conditioning the tonsil microenvironment with IL-9 or IL-33 would enable the rapid induction of durable and effective mucosal humoral immunity by the VC10014 DNA/protein HIV vaccine platform. Rhesus macaques were primed with VC10014 gp160 DNA plasmids in the absence or presence of IL-9 or IL-33 intra-tonsillar (IT), followed by intramuscular (IM) boosting with the DNA/protein HIV vaccine regimen. IT immunization with IL-9 or IL33 induced plasma Env-specific IgG that was present as early as week 6, mucosal Env-specific IgG and heterologous plasma neutralizing antibody. IL-33 treated animals exhibited the greatest number of germinal centers (GCs) and largest GCs following the first IM boost whereas IL-9 treated animals showed the greatest number of GCs following the second IM boost in the draining lymph nodes. The difference in kinetics, with IL-33 promoting an earlier B cell response, and IL-9 promoting greater magnitude later, may suggest that IL-9 and IL-33 promote the development of qualitatively different B cells subsets and may have synergistic potential in HIV vaccine development. Supported by a grant from NIH (R01DE027245).

Persistent immunogenicity of integrase defective lentiviral vectors delivering membrane-tethered native-like HIV-1 envelope trimers

Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.